Djordje Gveric

Astrocyte characterization in the multiple sclerosis glial scar

Dense astrocytic scarring in chronic multiple sclerosis (MS) plaques produces an inhibitory environment which can impede tissue repair. Animal studies have shown that the antigenic phenotype of the most abundant cell type in the brain, the astrocyte, varies depending on astrocyte type and location. To identify the phenotype of scar astrocytes (SAs) in chronic lesions, markers of reactive astrocytes characterized in animal studies were investigated. To date these are the only established markers. Cerebral subventricular deep white matter from normal control, MS normal appearing white matter and lesions (acute, subacute and chronic) were examined by immunohistochemistry and immunoblotting. The antigenic profile of SAs revealed significant modification of astrocyte protein expression in chronic MS lesions. SAs express nestin, embryonic neural cell adhesion molecule, fibroblast growth factor receptor 4, epidermal growth factor receptor, nerve growth factor and a subpopulation of SAs also express basic fibroblast growth factor. These are in addition to the expected markers glial fibrillary acidic protein, vimentin, and the tenascins C and R. Therefore, an SA antigenic phenotype has now been defined. This knowledge may allow the development of therapeutic strategies that prevent scar formation and promote tissue repair.

Attenuation of proliferation in oligodendrocyte precursor cells by activated microglia

Activated microglia can influence the survival of neural cells through the release of cytotoxic factors. Here, we investigated the interaction between Toll‐like receptor 4 (TLR4)‐activated microglia and oligodendrocytes or their precursor cells (OPC). Primary rat or N9 microglial cells were activated by exposure to TLR4‐specifc lipopolysaccharide (LPS), resulting in mitogen‐activated protein kinase activation, increased CD68 and inducible nitric oxide synthase expression, and release of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin‐6 (IL‐6). Microglial conditioned medium (MGCM) from LPS‐activated microglia attenuated primary OPC proliferation without inducing cell death. The microglial‐induced inhibition of OPC proliferation was reversed by stimulating group III metabotropic glutamate receptors in microglia with the agonist L‐AP4. In contrast to OPC, LPS‐activated MGCM enhanced the survival of mature oligodendrocytes. Further investigation suggested that TNF and IL‐6 released from TLR4‐activated microglia might contribute to the effect of MGCM on OPC proliferation, insofar as TNF depletion of LPS‐activated MGCM reduced the inhibition of OPC proliferation, and direct addition of TNF or IL‐6 attenuated or increased proliferation, respectively. OPC themselves were also found to express proteins involved in TLR4 signalling, including TLR4, MyD88, and MAL. Although LPS stimulation of OPC did not induce proinflammatory cytokine release or affect their survival, it did trigger JNK phosphorylation, suggesting that TLR4 signalling in these cells is active. These findings suggest that OPC survival may be influenced not only by factors released from endotoxin‐activated microglia but also through a direct response to endotoxins. This may have consequences for myelination under conditions in which microglial activation and cerebral infection are both implicated.

Insulin-like growth factors and binding proteins in multiple sclerosis plaques

Insulin‐like growth factors (IGFs) play an important role in development and myelination in the central nervous system (CNS) as well as in the proliferation and differentiation of cells of the immune system. To assess the influence of this growth factor family on demyelination and repair in multiple sclerosis (MS), the expression of IGF‐I, IGF‐II, insulin, IGF binding proteins (IGFBP) 1–3 and IGF‐I receptor (IGF‐IR) in CNS tissue from MS and normal control cases was studied by immunocytochemistry. In active MS lesions, the expression of IGF‐I, insulin and IGFBP1 was detected in hypertrophic astrocytes while that of IGF‐II and IGFBP2 and 3 was confined to foamy macrophages within lesions and activated microglia in adjacent white matter. IGF‐IR, the major IGF receptor, was immunolocalized in macrophages and an astrocyte subpopulation in plaques. Oligodendrocytes in normal‐appearing white matter expressed only IGFBP1, not IGFs or IGF‐IR. As the remyelinating capacity of oligodendrocytes could be impaired owing to the absence of IGF‐IR, the prevailing role of IGFs in inflammatory demyelination may be to promote phagocytosis of myelin and astrogliosis.

A role for the plasminogen activator system in inflammation and neurodegeneration in the central nervous system during experimental allergic encephalomyelitis

Early signs of inflammatory demyelination include entry of fibrin(ogen) into the central nervous system (CNS), which is normally excluded by the blood-brain barrier, and up-regulation of components of the plasminogen activator system. Using mice deficient in tissue-type plasminogen activator (tPA-/-) and urokinase plasminogen activator receptor (uPAR-/-), we investigated the involvement of the PA system on the clinical and pathological features of experimental allergic encephalomyelitis, an animal model of multiple sclerosis. tPA-/- mice suffered an early and a more severe acute disease characterized by incomplete recovery when compared to wild-type controls, with significantly higher CNS levels of plasminogen activator inhibitor-1. This correlated with fibrin accumulation, which co-localized with nonphosphorylated neurofilament on thickened axons in experimental allergic encephalomyelitis tissue. In contrast, uPAR-/- mice had a delayed, less acute disease reflected in delayed infiltration of inflammatory cells. These animals developed chronic disease as a result of steadily increased inflammation, increased levels of urokinase-type plasminogen activator (uPA), and greater degree of demyelination. Thus, the plasminogen activator system can modulate both inflammatory and degenerative events in the CNS through the respective effects of tPA and uPAR on fibrinolysis and cell adhesion/migration, manipulation of which may have therapeutic implications for multiple sclerosis.

Phosphorylation and compactness of neurofilaments in multiple sclerosis: Indicators of axonal pathology

Aims Axonal pathology extends to the axonal cytoarchitecture leaving its signature on axoskeletal proteins. This study investigated whether neurofilament (NfH) phosphorylation would relate to the dynamics of axonal pathology in multiple sclerosis (MS). Methods NfH phosphoforms (SMI32, SMI34, SMI35) were quantified by ELISA from microdissected samples of control and MS brain and spinal cord. Individual axons were analysed by electron microscopy, densitometrically and morphologically in adjacent tissue sections. Experiments were carried out pre- and post enzymatic dephosphorylation. Results In control tissue a rostro-caudal gradient of NfH indicated an increase in axonal density from the brain gray matter towards the spinal cord. The highest levels of phosphorylated and hyperphosphorylated NfH were found in acute lesions of brain and spinal cord, in contrast to chronic lesions where levels were lower than in white matter, consistent with axonal loss. Dephosphorylated NfH was higher, but less densly packed in MS white matter axons compared to control tissue. Conclusions The findings suggest that a less organised/compact axoskeleton or impaired axonal transport may represent an early sign of axonal pathology within the normal appearing white matter in MS. Subsequently a proportional increase of dephosphorylated NfH, aberrant phosphorylation and/or aggregation may occur whilst the protein is transported through the white matter towards the MS plaque, where hyperphosphorylated NfH dominates.

Production of IL-16 Correlates with CD4+ Th1 Inflammation and Phosphorylation of Axonal Cytoskeleton in Multiple Sclerosis Lesions

BackgroundMultiple sclerosis (MS) is a central nervous system-specific autoimmune, demyelinating and neurodegenerative disease. Infiltration of lesions by autoaggressive, myelin-specific CD4+Th1 cells correlates with clinical manifestations of disease. The cytokine IL-16 is a CD4+ T cell-specific chemoattractant that is biased towards CD4+ Th1 cells. IL-16 precursor is constitutively expressed in lymphocytes and during CD4+ T cell activation; active caspase-3 cleaves and releases C-terminal bioactive IL-16. Previously, we used an animal model of MS to demonstrate an important role for IL-16 in regulation of autoimmune inflammation and subsequent axonal damage. This role of IL-16 in MS is largely unexplored. Here we examine the regulation of IL-16 in relation to CD4+ Th1 infiltration and inflammation-related changes of axonal cytoskeleton in MS lesions.MethodsWe measured relative levels of IL-16, active caspase-3, T-bet, Stat-1 (Tyr 701), and phosphorylated NF(M+H), in brain and spinal cord lesions from MS autopsies, using western blot analysis. We examined samples from 39 MS cases, which included acute, subacute and chronic lesions, as well as adjacent, normal-appearing white and grey matter. All samples were taken from patients with relapsing remitting clinical disease. We employed two-color immunostaining and confocal microscopy to identify phenotypes of IL-16-containing cells in frozen tissue sections from MS lesions.ResultsWe found markedly increased levels of pro- and secreted IL-16 (80 kD and 22 kD, respectively) in MS lesions compared to controls. Levels of IL-16 peaked in acute, diminished in subacute, and were elevated again in chronic active lesions. Compared to lesions, lower but still appreciable IL-6 levels were measured in normal-appearing white matter adjacent to active lesions. Levels of IL-16 corresponded to increases in active-caspase-3, T-bet and phosphorylated Stat-1. In MS lesions, we readily observed IL-16 immunoreactivity confined to infiltrating CD3+, T-bet+ and active caspase-3+ mononuclear cells.ConclusionWe present evidence suggesting that IL-16 production occurs in MS lesions. We show correlations between increased levels of secreted IL-16, CD4+ Th1 cell inflammation, and phosphorylation of axonal cytoskeleton in MS lesions. Overall, the data suggest a possible role for IL-16 in regulation of inflammation and of subsequent changes in the axonal cytoskeleton in MS.

Myelin/Axonal Pathology in Interleukin-12 Induced Serial Relapses of Experimental Allergic Encephalomyelitis in the Lewis Rat

Lewis rats, on recovery from monophasic clinical experimental allergic encephalomyelitis (EAE), can be induced to develop repeated paralytic relapses with a graded reduction in clinical severity following intraperitoneal administration of IL-12. By the time of the third relapse, the number and size of inflammatory cuffs in the spinal cord were reduced with the makeup of the cellular infiltrate shifting to a significantly increased number of B cells. Serum levels of myelin basic protein (MBP)-specific IgG1 and IgG2b were found to rise over time while MBP and MBP peptide-positive macrophages and microglia became evident in perivascular cuffs and in spinal cord parenchyma, indicative of myelin phagocytosis. Axonal death was observed in semithin and EM sections of spinal cord in third relapse animals in association with iNOS and tPA immunostaining throughout gray and white matter. These neurotoxic or excitotoxic agents may contribute to axonal damage directly or indirectly by activated microglia and macrophages, leading to limited damage of the axonal-myelin unit.

Tenascin C induces a quiescent phenotype in cultured adult human astrocytes

Astrocytic scar formation occurs subsequent to brain and spinal cord injury and impedes repair. The exact mechanisms of scar formation have yet to be elucidated but it is known that astrocytes within the scar have a different antigenic phenotype from normal or reactive astrocytes. Astrocyte cell culture offers a suitable system to identify factors that induce the scar phenotype as well as factors that reverse this process and that may help identify therapeutic strategies to treat astrogliosis. However, when placed in standard culture conditions, astrocytes become activated/reactive and express molecules characteristic of scar tissue in vivo. In the present study, we made use of this phenomenon to identify culture conditions that change the activated phenotype of cultured astrocytes into one characteristic of normal quiescent astrocytes. In particular, we examined the effect of extracellular matrix (ECM) proteins found in the human brain, on the phenotype of human adult astrocytes. Significantly fewer astrocytes expressed scar properties when grown on tenascin‐C (TN‐C) than those cultured on other ECM proteins or poly‐L‐lysine‐coated dishes. TN‐C also significantly reduced the proliferation rate of the astrocytes in vitro. In addition, further manipulation of culture conditions induced partial astrocyte reactivation. Our findings suggest that astrocytes grown on TN‐C revert to a quiescent, nonactivated state that is partially reversible. This raises the possibility that therapeutic strategies aimed at manipulating TN‐C levels during CNS injury may help reduce astrocytic scarring.

tPA receptors and the fibrinolytic response in multiple sclerosis lesions

Axonal damage in multiple sclerosis (MS) lesions is associated with failure of fibrinolysis because of the inhibition of the plasminogen activator system. Plasma membrane receptors for tissue plasminogen activator (tPA) and plasminogen concentrate proteolytic activity on the cell surface and provide protection from inhibitors that in turn may locally enhance the fibrinolytic response. Therefore, we have investigated expression of two of these receptors in MS lesions, annexin II tetramer (AIIt) and low-density lipoprotein receptor-related protein (LRP). In acute MS lesions both AIIt and LRP were immunolocalized on macrophages and astrocytes while LRP was additionally found on neuronal cells in cortical gray matter. Western blot analysis confirmed a significant increase in AIIt in MS lesions and in a proportion of normal-appearing white matter samples, with a highly significant correlation between annexin II levels and factors associated with impeded fibrinolysis, such as plasminogen activator inhibitor-1. Immunoblotting analysis of plasmin(ogen) revealed increased levels of lysine-plasminogen in samples expressing high AIIt protein levels. Our results suggest that limited availability of tPA in MS lesions because of formation of tPA-plasminogen activator inhibitor-1 complexes reduces capability of tPA receptors to generate plasmin, which further diminishes fibrinolytic capacity in active MS lesions and possibly leads to axonal damage.

Myelin phagocytosis and remyelination of macrophage‐enriched central nervous system aggregate cultures

An increased level of myelin basic protein (MBP) degradation peptide 80–89, representative of myelin breakdown, is detected in myelinating foetal rat brain aggregate cultures supplemented with peritoneal macrophages at a time coinciding with the onset of myelination. During the period of myelination, the proportion of activated macrophages/microglia in the aggregates decreases, accompanied by a reduction in the content of MBP degradation products. During the recovery period following a demyelinating episode, the rate of MBP synthesis in antibody‐treated standard aggregates was greater than in their medium controls. However, the rate of MBP accumulation was not as efficient in macrophage‐enriched aggregates and was associated with persistently raised MBP peptide levels. Thus, as occurs in multiple sclerosis lesions, attempts at remyelination appear to be counterbalanced by macrophage‐mediated demyelination, with the continued presence of degraded myelin rendering a local environment that is not fully conducive to remyelination. J Neurosci. Res. 66:1173–1178, 2001.